The aim of this proposal is to elucidate functional aspects of nitrogenase, specifically the mechanism of substrate activation and reducton by the enzyme. The proposed work will: 1) better define molecular criteria, such as steric and electronic effects, for binding of reduicible substrates at the active site of nitrogenase; 2) provide more detailed information on the processes of electron transfer andproton transfer to substrate, including reduction stereochemistry; 3) probe the existence andprobable nature of nitrogen reduction intermediates on the catalytic pathway, with particlular reference to diazene-like intermedites; 4) elaborte chemical aspects of nitrogenase reductons to obtain mechanistic information and also define useful criteria for evaluating functional homology between the enzyme and biomimetic model systems, and between the enzyme and tis FeMo-cofactor; 5) help unravel relationships among different nitrogenase substrates, and investigate extrinsic effects on their reductions by the enzyme; 6) create a foundation for the development of affinity labels of the active site. The methodology is based on the use of novel substrtae analogs, such as cyclopropene, fluorinated cyclopropenes, diazirine (cyclodiazomethane) and other diazene derivatives. Substrate interactions with nitrogensase will be characterized using product analysis, kinetic techniques and inhibiton studies. Deuterium isotope methods will be utilized to determine reduction stereochemistries. Implementation of the proposed research will provide new insights into the mechanism of nitrogenase-catalyzed reductions.